Previously, we had constructed an ensemble of chimeric G-alpha subunits using G-alpha-i1 as the backbone and introducing sequence from G-alpha-q to test for the sequences within G-alpha conferring receptor-selectivity. These studies revealed that the receptor contact surface of the G-alpha subunit comprises an extensive region of at least 50 amino acid residues of the carboxyl terminus of the G-alpha-q. We were limited in those investigations by inability to express chimeric alpha subunits with additional sequence contributed by G-alpha-q. To determine if these results apply to other G-protein types and to examine for additional sequence(s) specifying selective contact, we have constructed a similar set of chimeric structures introducing sequence from the G-alpha-s protein into G-alpha-i1 and testing for interaction with the beta2-adrenergic receptor. We were able in these studies to replace the majority of the sequence of G-alpha-i1 with G-alpha-s derived sequence and still obtain expression of functional G-alpha protein. These studies with the beta2-adrenergic receptor and G-alphapi1/s chimeras recapitulated the results found previously for G-alpha-i1/q chimeras and the 5HT2c receptor. Further, they rule out all other G-alpha-s structure save the N-terminal alpha helix as providing receptor-selective contact (Gutierrez et al, manuscript in preparation). In collaboration with Dr. Reinhard Grisshammer, NINDS, we have continued efforts to obtain crystals of GPCR-G-protein complexes using cephalopod rhodopsin and G-alpha-q or G-alpha-t. Do date all experiments have failed to yield crystals of the complexes. Neither have we succeeded in obtaining crystals of the activated cephalopod rhodopsin. We initiated a project in collaboration with Dr. John Tesmer, University of Michigan School of Medicine, to obtain crystals of the complexes of cephalopod rhodopsin with G-receptor kinase 2 with and without G-alpha-q. These have also failed to yield crystals to date. Also in collaboration with Dr. John Tesmer, we have succeeded in obtaining the crystal structure at 2.0 angstrom resolution of a 90 kDa fragment of the cephalopod retinal phospholipase C. Our structure reveals a previously unkown interdomain interaction between C-terminal sequence of the PLC acting as an auto-inhibitor of the catalytic domain. This structure suggests a novel mechanism for regulation of PLC-beta enzymes by G-alpha-q in which the G-protein releases the PLC from auto-inhibition (Lyon et al, manuscript in preparation).